Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or "modular" and Type II "iterative" PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Modular PKS enzymes for 14-membered polyketides are encoded by PKS genes that often consist of three or more open reading frames (ORFs). Each ORF of a modular PKS can comprise one, two, or more "modules" of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or "domains." These large multifunctional enzymes (&gt;300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying .beta.-carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a "clean" host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of .beta.-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
Oleandomycin is an antibacterial polyketide (described in U.S. Pat. No. 2,757,123, incorporated herein by reference) produced by a modular PKS in Streptomyces antibioticus. Oleandomycin has the structure shown below, with the conventional numbering scheme and stereochemical representation. ##STR1##
As is the case for certain other macrolide antibiotics, the macrolide product of the PKS, 8,8a-deoxyoleandolide, also referred to herein simply as oleandolide (although oleandolide in other contexts refers to the epoxidated aglycone), is further modified by epoxidation (at C-8 and C-8a) and glycosylation (an oleandrose at C-3 and a desosamine at C-5) to yield oleandomycin.
The reference Swan et al., 1994, entitled "Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence," Mol. Gen. Genet. 242: 358-362, incorporated herein by reference, describes the DNA sequence of the coding region of a gene designated ORFB hypothesized to encode modules 5 and 6 and a fragment of a gene designated ORFA hypothesized to contain the ACP domain of module 4 of the oleandolide PKS. The reference Quiros et al., 1998, entitled "Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus," Mol. Microbiol. 28(6): 1177-1185, incorporated herein by reference, describes genes and gene products involved in oleandomycin modification during its biosynthesis. In particular, the reference describes a glycosyltransferase involved in rendering oleandomycin non-toxic to the producer cell and a glycosidase that reactivates oleandomycin after the glycosylated form is excreted from the cell. See also Olano et al., August 1998, "Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308, and PCT patent publication No. 99/05283, incorporated herein by reference. While a number of semi-synthetic oleandomycin derivatives have been described, see U.S. Pat. Nos. 4,085,119; 4,090,017; 4,125,705; 4,133,950; 4,140,848; 4,166,901; 4,336,368; and 5,268,462, incorporated herein by reference, the number and diversity of such derivatives have been limited due to the inability to manipulate the PKS genes.
Genetic systems that allow rapid engineering of the oleandolide PKS would be valuable for creating novel compounds for pharmaceutical, agricultural, and veterinary applications. The production of such compounds could be accomplished if the heterologous expression of the oleandolide PKS in Streptomyces coelicolor and S. lividans and other host cells were possible. The present invention meets these and other needs.